Product containing growth factor and glutamine and use of growth factor for the treatment of intestinal mucosa

ABSTRACT

IGF-1 is administered to a patient for the treatment or prevention of atrophy of the gut mucosa.

The present invention relates to a product having anabolic effects andwhich also can be used for the treatment or prevention of malfunction ordiseases of the intestinal mucosa. The product contains a growth factortogether with glutamine and a fatty acid. The invention also comprises aprocedure for the prevention or treatment of impaired function of thegut wall after, for example, trauma, sepsis, burn injuries, radiationtherapy or cytotoxic chemotherapy, as well as different inflammatoryconditions such as ulcerative colitis or morbus Chron. Thereby cangrowth factors optionally together with glutamine and/or a fatty acid begiven by oral, intravenous, peritoneal, rectal, intramuscular orsubcutaneous administration.

BACKGROUND

In trauma and sepsis, in addition to the conspicuous injuries or signsof infection, there are also metabolic changes. These changes can bedivided into an early so-called ebb phase followed later by a so-calledflow phase. In the ebb phase (shock phase), which can last for 1-3 daysafter the incidence of the injury, there exists a reduced energyturnover, increased lipolysis, hyperglycaemia, elevated catecholaminerelease and hormonal maladaptation, circulatory effects with decreasingblood pressure, reduced tissue perfusion and oxygen consumption,hypoxia, acidosis, increased body weight as a result of fluid and urineretention and a reduced body temperature. Protein synthesis is alsoreduced as well as blood albumin and amino acid concentrations.

After about 3 days, the flow phase appears which can last for 1-4 weeks.The flow phase is characterised by an increased energy turnover,increased release of substrates, negative nitrogen balance, rise inblood pressure, increased oxygen consumption and elevation of bodytemperature. Protein synthesis is reduced in mild trauma whilst bothsynthesis and breakdown increase in major trauma. Breakdown dominatesresulting in a negative nitrogen balance which can reach 0.3-0.6 gnitrogen per kg body weight and day. This is equivalent to approximately1.2-2.4 kg muscle wastage per day in a normal individual.

If the catabolic component of the flow phase continues for too long, alife threatening situation can develop with substantial losses of fatand muscle tissue as well as impaired wound healing and defence againstinfection (Ljusk S Eriksson, Mossberg T, Wahren J; pp 99-117, Klin NutrAlmq & Wiks 1987).

The concentration of glycogenic amino acids (such as alanine, glycine,serine and glutamic acid) drops in the plasma whilst a simultaneousincrease in the plasma levels of branched chain amino acids (such asleucine, isoleucine and valine), aromatic amino acids (such asphenylalanine, tyrosine and tryptophan) as well as methionine, isobserved. An approximately 50% reduction of the intracellular glutamineconcentration is also typical. The above mentioned changes can partly beexplained as disturbances in transport over the cell membrane. Alanineand glutamine are taken up and metabolised by the gut mucosa and theliver. In the liver they are utilised for, amongst other things,gluconeogenesis. In burn patients the splanchnic (liver and gut) uptakeof amino acids is increased approximately 2-6-fold compared to a healthycontrol. The protein catabolism, which is evident mainly as increasedurea production, not only affects striated muscles and visceral proteinsbut also smooth muscles such as those found in the gut wall and in therespiratory pathways. This situation can negatively affect vital bodilyfunctions as well as secretory transport in the bronchii, with secretorystagnation and development of pneumonia, a common post-traumaticcomplication.

The gut mucosa is important not only for the selective and activeabsorption of different nutrients, but also as a barrier against foreignorganisms and toxins. In recent years, it has been shown that thebarrier function of the gut mucosa deteriorates in association withmajor trauma and sepsis. Furthermore, both cytotoxic chemotherapy andradiation therapy have a deleterious effect on gut mucosal function.Intravenous nutritional therapy, which is applied when the patient hasdifficulties to eat or assimilate orally administered food, has beenshown to contribute to atrophy of the gut mucosa. Gut atrophy appears inlaboratory animals and in man after intravenous feeding. This canincrease the risk for translocation of bacteria over the gut mucosa withconsequent risk for sepsis and ,increased mortality.

Atrophy of the gut combined with simultaneous translocation of bacteriaover the gut mucosa is most likely in patients who have suffered majortrauma and extensive clinical intervention, in sepsis, in major burninjuries and even after radiation therapy and/or cytotoxic chemotherapyin patients with abdominal or urogenital tumours. Patients withinflammatory diseases of the gut such as ulcerative colitis and morbusChron also run the risk of being stricken by life threatening infectionscaused-by atrophy of the villi and consequent development of ileus withtoxic dilatation and release of bacteria into the peritoneum.

The observation of a marked decline in the intracellular concentrationof glutamine in muscle following trauma and sepsis initiated interest inglutamine metabolism and its significance. It has been shown thatglutamine is an important energy substrate in intestinal tissue and thatglutamine utilization increases in the catabolic state. In studies onexperimental animals it has been demonstrated that glutamineadministration can prevent a fall in plasma and muscle glutamineconcentrations following trauma. Further, an accelerated mucosa recoveryafter injury induced by 5-fluorouracil was observed in rats whenglutamine was added to the intravenous nutrition solutions ('Dwyer ST etal, Clin Res 367a, 1987).

During an international symposium "Glutamine metabolism in health anddisease", Jan. 26-27, 1990, in San Antonio, Tex., USA, it was reportedthat in human subjects, post-operative glutamine supplementation of atotal parenteral nutritional regimen improved nitrogen balance andcounteracted the decline in protein synthesis usually seenpost-operatively. Further it was reported that a number of positiveeffects had been observed in patients undergoing bone marrowtransplantation who were given considerable amounts of glutamine.

In a patent for the treatment of catabolic dysfunction in an animal, acomposition including 5-30 g of L-glutamine and/or 5-25 g of alpha-ketoglutaric acid is claimed (WO 87/01589). Another patent comprises aqueoussolutions for parenteral nutrition which preferentially containglutamine together with other organic nitrogen-containing compounds (WO87/03806). Furthermore, the presentation of glutamine in the form ofdipeptides has been patented (see EP 87750 and DE 3108079).

The gut has an active endogenous microflora of which the majority of theorganisms are facultative of obligatory anaerobes. This means that themajor products of fermentation are the short-chain fatty acids (SCFA),mainly acetic acid, propionic acid and butyric acid. The substrates forthis fermentation are the carbohydrates that reach the large intestine.

In so-called germ-free rats there is an atrophy of the gut mucosa. Theserats also have a low endogenous production of SCFA which represents onlyabout 1% of the production seen in normal rats (Hoverstedt andMidtvendt, J. Nutr. 116, 1772-76, 1986).

A fiber-free diet (one week) or total parenteral nutrition (two weeks)reduces the fecal loss of SCFA in man by 50% and 80%, respectively(Hoverstedt, Symposium, held at The Wennergren-Gren Center, Stockholm,Sweden, Jun. 1th-4th, 1988).

Some experiments with SCFA-enriched nutritional solutions have beendescribed in the literature. In all cases but one the experiments werecarried out in animals that had been subjected to small bowel resectionand given intracolonal or intracaecal infusions.

In a study of Koruda et al. (Am. J. Clin. Nutr. 51,685-9, 1990)SCFA-enriched total parenteral nutrition was employed, but since therewas no fat in the regimen, the diet was not balanced (lacking essentialfatty acids). Furthermore, a mixture of SCFA (acetate, propionate andbutyrate) was used in which acetate and propionate were in higherconcentration than butyrate. These shorter acids have been shown to bemore toxic than butyrate.

It has been suggested (WO 87/04074) that protein accretion or nitrogenretention can be promoted in the case of a hypocaloric diet by theadministration of growth hormone. In a study with intravenously fedrats, an increase in mucosal protein was observed in the small bowel andin the colon following the simultaneous administration of L-glutamineand epidermal growth factor (Jacobs D. O. et al. Surgery 104 (2),358-364 (1988)). IGF-I has been shown by O'Sullivan et al.(Endocrinology 125: 2793-95 81989) to prevent weight loss in starvedmice, whereas GH (bovine) was ineffective in this situation. A relativeincrease in kidney and spleen weights has been reported for GH and IGF-1treated rats ( Skottner A. et al. Endocrinology 124, 2519 (1989).

It is quite clear, however, that several of the problems mentioned abovehave until now not been solved with existing nutritional therapy.

At present there is no clinical application of a growth factor alone ortogether with glutamine and/or a short- or medium-chain fatty acid forthe improvement of gut function or for the combination having anaboliceffects.

DESCRIPTION OF THE INVENTION

The present invention relates to a product containing glutamine or aderivative or analogue thereof, a short- or medium-chain fatty acid anda growth factor or analogue thereof for simultaneous, separate orsequential use having anabolic effects and/or for treatment orprevention of malfunction or disease of the intestinal mucosa as well asthe use of these components for the manufacture of a medicament or a kitfor the treatment or prevention of malfunction or disease of theintestinal mucosa.

The invention also discloses a method for the treatment or theprevention of malfunction or disease of the intestinal mucosacharacterised in that a pharmacologically effective dose of a growthfactor or analogue thereof is administered to a patient optionallysimultaneously, separately or sequential with either or both ofglutamine or a derivative or analogue thereof and/or a short- ormedium-chain fatty acid.

The components should be in a pharmaceutically acceptable form. Byproduct is meant here not only a product or kit containing these threecomponents, but also when the components are admixed with othercomponents in a TPN, such as amino acids, carbohydrates and fat,preferably as an emulsion.

The components can also be administered separately, e.g. a short -ormedium- chain fatty acid, glutamine and the growth factor in an aqueousmedium. The fatty acid and glutamine can both be mixed with thenutritient and the growth factor administered separately. Glutamine canbe administered e.g. as the L-amino acid, as N-acetyl-L-glutamine or asa glutamine-containing peptide.

By short-or medium-chain fatty acids are meant here saturated fattyacids of C2-C12 carbon chain length, preferably short chain of 2-4carbons, such as butyric acid which is preferably administeredintravenously.

The short- or medium-chain fatty acids can be administered as, forexample, sodium or potassium salts and a medium fatty acid can beadministrated as a triglyceride.

Growth factors include here especially growth hormone (GH), Insulin-likegrowth factors IGF-1 and IGF-2, GRF (growth hormone releasing factor) oranalogue thereof etc. By analogue is meant substances which have thesame physiological effect as the Growth factors, mainly I GF-1, can beinjected or can be administered intravenously either as an aqueoussolution or incorporated into a fat emulsion or as a component inmicelles or liposomes.

The amount of the components given to humans could be varied dependingon the patient and disease. Normally they are within the doses normallygiven, such as 0.01-1 g/kg, day for glutamine, preferably 0,2-0,7 g/kg,day and 0,005-0,3 g/kg per day for fatty acid, preferably 0,02-0,15 g/kgper day. The amount of IGF-1 could be 0.02-20 mg/kg per day andpreferably 0.05-2 mg/kg per day, and for GH up to 1 IU/kg per day,preferably 0.05-0.7 IU/kg per day. (4 IU GH is equal to 1.3 mg GH).

As can be seen from the background and description of the problem, thefunction of the gut mucosa can be of critical importance in the outcomeof different illnesses. In a series of experiments, mainly in the rat,we have studied the effect on the gut mucosa of intravenousadministration of commercially available solutions (for example, aminoacid solutions, glucose solutions and fat emulsions) both with andwithout supplementation with glutamine, short-chain or medium-chainfatty acids and growth factors.

The measured variables have been primarily growth, gut mucosamorphology, villus height, crypt depth and cell proliferation in the gutmucosa measured with a radiolabelled thymidine incorporation technique.

In the above mentioned studies we observed a pronounced atrophy of thegut mucosa after total parenteral nutrition. As expected we noted a 30%reduction in the villus height and crypt depth compared to an orally fedcontrol group. Addition of glutamine improved the gut mucosa; villusheight and crypt depth increased by about 20 % and a similar improvementwas seen after the administration of either a short-chain fatty acid(mainly butyric acid) or IGF-1.

When glutamine, butyric acid and IGF-1 were administered together, anunexpected synergistic effect was observed on both growth and the gutmucosa. Growth rate was three-fold higher than that of rats infused withfat, amino acids and glucose, which was totally unexpected and ananabolic effect was thus clearly shown. Moreover, for the first time wecould reach a villus height in the TPN-fed rat which approached 400 μm(compared to slightly over 300 μm after non-supplemented infusion). Thisfinding was confirmed by the results of the mucosal cell proliferationstudies.

EXAMPLE 1

Male Sprague Dawley rats (180-190 g body weight, BW) were surgicallyfitted with a permanent central catheter which was protected by aharness. Total parenteral nutrition was given to the rats in the form ofa standard regimen:

    ______________________________________                                        Fat              7.6    g/kg BW and day                                       Nitrogen         1.35   g/kg BW and day                                       Total energy intake                                                                            270    kcal/kg BW and day                                    Total volume intake                                                                            300    ml/kg BW and day                                      ______________________________________                                    

The ratio between glucose and fat in this experiment was 30/70 expressedas a % of the non-protein energy intake. The TPN (total parenteralnutrition) mixtures were obtained by mixing Vamin® 14 EF (a solution ofessential and non-essential amino acids for parenteral nutrition), a 50%glucose solution and Intralipid® 20% in relevant proportions after whichwas followed by addition of the necessary amounts of electrolytes, traceelements and vitamins required by the rat.

On the morning of the first experimental day the rats were randomisedinto three groups: oral controls, intravenous controls and the glutaminegroup. The oral control rats received a standard diet (R3, Ewos AB,Sodertalje, Sweden) ad libitum during the experimental period and theintravenous control rats received the above-described standard TPNregimen.

The glutamine group received glutamine in the form of the dipeptideglycyl-glutamine (which was administered intravenously in isonitrogenousand isocaloric amounts at the expense of the amino acid solution) aswell as the other nutrients supplied according to the regimen given tothe intravenous control group.

The infusions were given for 7 days and growth was recorded daily. Thetotal weight gain was 41.4±4.2, 14.9±3.2 and 8.6±3.5 for the oralcontrols, intravenous controls and glutamine groups, respectively.

After 3 hours' fasting, blood samples were taken under pentobarbitalanaesthesia for analysis of the clinical chemistry. The results areshown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Clinical chemistry analyses in the serum.                                     Group  ASAT    ALAT    ALP  LD   Bilirub                                                                             Chol betaHB                            ______________________________________                                        Oral   1.4     0.87    8.2  3.6  0.7   2.0  1.3                               control                                                                       iv.    1.3     0.37    4.4  5.8  1.7   2.8  1.1                               control                                                                       Glu-   1.3     0.44    5.0  3.5  1.6   2.5  1.1                               tamine                                                                        ______________________________________                                    

Abbreviations: ASAT=aspartate aminotransferase(microkat/1); ALAT=alanineaminotransferase (microkat/1): ALP=alkaline phosphatase microkat/1);LD=lactate dehydrogenase (microkat/1):; Bilirub=Bilirubin (micromol/1);Chol=cholesterol (mmol/1); β-HB=β-hydroxy butyrate (mmol/1).

The rats were sacrificed by exsanguination after heart puncture underpentobarbital anaesthesia after which obduction and macroscopicexamination was performed. After sacrifice the liver, spleen, lungs,kidneys, heart and thymus were dissected out and weighed. The relativeorgan weights showed no differences of biological relevance between thegroups. Similarly, there were no marked differences in the bloodchemistry.

Sections of the gut were fixed in a Milla-Pott fixation medium and wereevaluated with respect to villus height and crypt depth (Table 2).

                  TABLE 2                                                         ______________________________________                                        Morphometrical evaluation of the jejunum.                                               jejunum                                                             Group       crypt depth (μm)                                                                        villus height (μm)                                ______________________________________                                        Oral control                                                                              165          460                                                  iv. control 121          309                                                  Glutamine   145          378                                                  ______________________________________                                    

The morphometric analysis of the jejunum demonstrates an atrophy of thegut after parenteral nutrition of the rats for 7, days. This conditioncould be markedly improved by the administration of glycyl-glutamine.

In summary, the results show that intravenous administration ofglutamine for 7 days was well tolerated and that a significantimprovement of gut morphology, with approximately a 20% increase in bothvillus height and crypt depth.

EXAMPLE 2

In an experiment performed similar to example 1, the effect of butyricacid on body weight, relative organ weight, blood chemistry, serum andurine β-hydroxy butyric acid levels as well as morphometrical evaluationof the jejunum was studied in rats. TPN was given to the rats in theform of the standard regimen described in example 1 and butyric acid wasgiven intravenously as Na-salt.

Infusions were given for 7 days and the growth of the animals wasstudied during the entire experimental period (Table 3).

                  TABLE 3                                                         ______________________________________                                        Growth after 7 days of TPN in the rat                                         Group         Weight increase (g)                                             ______________________________________                                        iv. control   14.9 ± 3.2                                                   Butyric acid  15.5 ± 2.5                                                   ______________________________________                                    

Blood sampling, sacrifice and dissection were performed as described inexample 1.

Total weight increase was not significantly different. There were alsono differences in the relative organ weights between the groups.Similarly, there were no differences in the blood chemistry.

Morphometric analysis of the jejunum and ileum demonstrated an atrophyof the gut after 7 days of parenteral nutrition in the rat. Thiscondition was improved by the administration of 3 g butyric acid per kgbody weight and day (table 4).

                  TABLE 4                                                         ______________________________________                                        Morphometric evaluation of the jejunum after 7 days of                        TPN in the rat.                                                               Group       crypt depth (μm)                                                                        villus height (μm)                                ______________________________________                                        iv. control 121          309                                                  Butyric acid                                                                              150          361                                                  ______________________________________                                    

In summary, the results show that 7 days of TPN containing 3 g/kgbutyric acid was well tolerated and that an improvement of gutmorphology as a consequence of butyric acid could be seen.

EXAMPLE 3

The effect of the simultaneous administration of glutamine, butyric acidand IGF-1 was studied in the rat. The experimental design as well as theparameters under study were identical to those described in examples 1and 2. Besides a morphometric evaluation of the small intestine, acytokinetic study using methyl-3-H thymidine was performed on thejejunum. The test group received parenteral fat, amino acids, glucose,butyric acid and glutamine. Further, the rats received IGF-1 in the formof subcutaneous injections of 1 mg/day divided into two doses each day.This was repeated for 8 days. At the same time an intravenous controlgroup of TPN rats was studied.

The experiment was terminated by anaesthetising the rats withpentobarbital and infusing them with radiolabelled methyl-3-H-thymidine(1 μC/kg). The rats were sacrificed by cervical dislocation and sampleswere taken from the jejunum for mophometric and cytokinetic analyses.See Table 5. Blood samples were taken for IGF-1 determination.

                  TABLE 5                                                         ______________________________________                                        Weight gain and morphometric evaluation of the jejunum                        after 7 days of TPN in the rat.                                                               jejunum                                                                             crypt depth                                                                             villus height                                 Group    Weight gain  (μm)   (μm)                                       ______________________________________                                        iv. control                                                                             8.8 ± 1.8                                                                              121       309                                           combination                                                                   therapy  27.1 ± 4.6                                                                              144       395                                           ______________________________________                                    

In summary, after the simultaneous administration of a short-chain fattyacid, glutamine and a growth factor IGF1, a surprising and significantsynergistic effect was observed both on growth and on the gut mucosa.

The effect on weight increase when the three components were giventogether was surprisingly high when compared to the weight increase fora component given alone (Example 1 and 2). Thus an anabolic effect wasobtained.

The effect on the gut mucosa was also clear and could even be seen atdissection. The results of the cytokinetic studies confirm theseobservations.

Thus, our studies show a surpringsingly good effect on the gut mucosawhen the three components were administered simultaneously, as comparedto any of the components given alone.

What is claimed is:
 1. Method for the treatment of atrophy of the gutmucosa after intravenous nutrition, which comprises injecting a patientin need thereof with a pharmacologically effective dose of IGF-1.
 2. Themethod of claim 1 wherein said dose is 0.02-20 mg/kg/day.
 3. The methodof claim 1 wherein said dose is 0.05-2 mg/kg/day.
 4. The method of claim3 wherein a mixture of glutamine, butyric acid and IGF-1 is injected. 5.The method of claim 1 which comprises improving the villus height of thegut.
 6. The method of claim 1 which comprises improving the crypt depthof the gut mucosa.
 7. The method of claim 1 which comprises improvingthe growth of the gut mucosa.
 8. The method of claim 1 wherein saidinjecting is intravenously.
 9. Method for the treatment of atrophy ofthe gut mucosa characterized in that a pharmacologically effective doseof IGF-1 thereof is injected into a patient simultaneously, separatelyor sequentially with either or both of:a) at least one member selectedfrom the group consisting of glutamine, N-acetyl-L-glutamine andglutamine-containing peptide; b) short-or-medium chain fatty acidcontaining 2-12 carbon atoms.
 10. The method of claim 9 wherein theamount of said glutamine is sufficient to provide a dose of 0.01-1g/kg/day, the amount of said butyric acid is sufficient to provide adose of 0.005-03 g/kg/day, and the amount of IGF-1 is sufficient toprovide a dose of 0.02-20 mg/kg/day.
 11. The method of claim 9 whereinthe amount of said glutamine is sufficient to provide a dose of 0.2-0.7g/kg/day, the amount of said butyric acid is sufficient to provide adose of 0.02-0.15 g/kg/day, and the amount of said IGF-1 is sufficientto provide a dose of 0.05-2 mg/kg/day.
 12. The method of claim 9 whereinsaid injecting is intravenously.
 13. The method of claim 9 whichcomprises improving the villus height of the gut.
 14. The method ofclaim 9 which comprises improving the crypt depth of the gut mucosa. 15.The method of claim 9 which comprises improving the growth of the gutmucosa.